Otitis media (OM) is the most common childhood infection and also the leading cause of conductive hearing loss in children. While it has been shown that overproduction of mucin, the major protein of mucus in the middle ear, plays an important role in the development of conductive hearing loss, little is known about the molecular mechanisms underlying mucin overproduction. Our long- term goal is to understand the molecular mechanisms by which mucin is up-regulated in OM. Based on our recent studies showing up-regulation of mucin transcription by Gram negative bacterium Pseudomonas aeruginosa in cystic fibrosis and that nontypeable Haemophilus infuenzae (NTHi) is known to be a major Gram negative bacterial pathogen in OM, we hypothesize that NTHi surface molecules up-regulate mucin gene transcription via activation of specific signaling pathways in middle ear epithelial cells. Recent advances in cloning promoter of mucin MUC5AC gene and establishing middle ear epithelial cell lines offer the opportunity to address the proposed hypothesis at molecular and cellular levels. Our preliminary results indicate that NTHi outer membrane proteins (OMPs) up-regulate mucin MUC5AC transcription via activation of a p38 MAP kinase signaling pathway. These encouraging results have thus laid a solid foundation for further investigation of the molecular mechanisms underlying NTHi-induced mucin transcription. Aim 1. Identify bacterial surface molecules responsible for mucin induction by purifying NTHi surface OMPs using chromatography and testing the fractions and proteins for mucin-inducing ability using luciferase assay. Aim 2. Determine the major intracellular signaling pathway required for NTHi-induced mucin transcription by using specific inhibitors and overexpressing dominant-negative mutants. Aim 3. Identify the NTHi response elements in the mucin MUC5AC gene promoter region and the cognate transcription factors by mutagenesis of MUC5AC promoter, using luciferase assay and gel mobility shift assay. Significance: These studies will provide new insights into the molecular pathogenesis of OM and open up new therapeutic targets for inhibition of mucus overproduction to prevent conductive hearing loss and recurrent infection in OM.